Effective isolation and collection of rare cells from a heterogeneous cell population remains of high interest, due to the increasing demand for isolated cell populations for use in disease diagnosis and treatment, e.g. gene therapy, as well as for basic scientific research. For example, pathologically changed cells, such as cancerous cells, can be separated from a larger normal cell population, and the cleaned cell populations may then be transplanted back into the patient.
One prominent demand is for the isolation of fetal cells to permit early fetus diagnosis, such as early screening of potential chromosomal disorders during pregnancy; fetal cells have been obtained by methods such as amniocentesis or chorionic villus sampling. Although one using such methods can obtain a significant amount of fetal cells such as to permit reliable diagnosis, such methods pose risks, especially to the fetus, because they require invasion into the uterus and typically can only be done after the first trimester.
Fetal cells are also present in circulating maternal blood as these cells pass from fetus to the maternal bloodstream in very low numbers; however, this results in ratios of fetal cells to maternal cells on the order of only a few ppm. In principle, a sample of the maternal blood obtained by venipuncture may be used for fetal diagnosis; however, there are some significant challenges associated with such a method to isolate and collect the rare fetal cells from the major population of maternity cells. These challenges also exist in separating fetal cells from cervical mucosa, and they may also be common to other rare cell recoveries from bodily fluids or the like, as well as to the separation and isolation of other biomolecules present in only minute quantities.
Cell separation is a rapidly growing area of biomedical and clinical development, and improved methods of separating a desired cell subset from a complex population will permit a broader study and use of cells that have relatively uniform and defined characteristics. Cell separation is also widely used in research, e.g. to determine the effect of a drug or treatment on a targeted cell population, to investigate biological pathways, to isolate and study transformed or otherwise modified cell populations; etc. Present clinical uses include, for example, the isolation of hematopoietic stem cells for reconstitution of blood cells, particularly in combination with ablative chemo- and radiation therapy.
Cell separation is often achieved by targeting molecules on the cell surface with specific affinity ligands in order to achieve selective, reversible attachment of a target cell population to a solid phase. In a subsequent step, nonspecifically adsorbed cells are removed by washing, followed by the release of target cells. Such specific affinity ligands may be antibodies, lectins, receptor ligands, or other ligands that bind proteins, hormones, carbohydrates, or other molecules with biological activity.
One of the current methods used to recover rare cells from a major bystander population is to use polystyrene macrobeads coated with an antibody selective for the target cell population. Such macrobeads coated with specific antibodies are often allowed to settle by gravity down through a suspension of a heterogeneous cell population so that macrobeads capture target cells by interception.
There are also other substrates used with columns to separate the target cells from a sample fluid, and generally, the type of substrate selected for performing the separation will determine how the targets are ultimately separated from the sample fluid. A substrate is generally provided with characteristics such that the desired targets will have substantially different binding propensities to the substrate than will the remaining components of the sample. An example of a column-type apparatus for cell separation is found in U.S. Pat. No. 5,240,856, issued Aug. 31, 1993, where the cells bind to a matrix within the column. In U.S. Pat. No. 5,695,989, issued Dec. 9, 1997, a column is designed to serve as a pliable vessel which can be squeezed to facilitate removal of bound cells. U.S. Pat. No. 5,672,481, issued Sep. 30, 1997 describes an apparatus for separation in a closed sterile field, where a single rigid vessel is used for collection, concentration and transfer. U.S. Pat. No. 5,763,194 describes a cell separation device comprised of an array of semi-permeable hollow fibers, where ligands are attached to the inner surface.
A number of problems and technical difficulties are associated with the above-mentioned approaches and also with the fairly widely used, macrobead-capture approach, which is frequently referred to as the column method as it basically utilizes macrobeads settling through a column. One major hurdle yet to be cleared to make the column method widely practical is the difficulty with non-specific binding. Moreover, using macrobeads to capture target cells in such a column separation method requires the recovery of the beads from the fluid stream and then cleaning, the usual procedure for which is washing. Beads may be subject to loss, collapse or aggregation during washing, leading to a less efficient collection of the target cells.
Capture agents, often antibodies selective for the particular target cell population, are generally physically or chemically attached to the bead surface. Capture agents which are physically attached to beads may fall off or be displaced as a result of transfer and handling. However, if they are attached strongly through covalent bonds so that the captured cells will definitely stay on the beads during washing, they may not thereafter be released and recovered during collection.
In addition to the column separation method, other methods have now been developed for separating target cells from a diverse population of cells such as may be found in bodily fluid or the like. For example, U.S. Patent Publication No. 2004/0142463 teaches systems for separating fetal cells from maternal blood or the like where undesired components from the blood sample are first separated using surfaces of plates or other solid supports, including columns, which carry specific binding members; then electrophoresis or the like is used to complete the separation and analysis of the target cells.
Published U.S. Patent Application No. 2004/038315 attaches releasable linkers to the interior luminal surfaces of capillary tubing, with the desired bound cells subsequently being released via a cleavage reagent and recovered. U.S. Published Patent Application No. 2002/132316 uses microchannel devices to separate cell populations through the use of a moving optical gradient field. U.S. Pat. No. 6,074,827 discloses the use of microfluidic devices that are constructed to have “enrichment channels” wherein electrophoresis is used to separate and identify particular nucleic acids from samples. Also mentioned is the optional use of antibodies or other binding fragments to retain a desired target biomaterial. U.S. Pat. No. 6,432,630 discloses a microflow system for guiding the flow of a fluid containing bioparticles through channels where selective deflection is employed, and it indicates that such systems may be used to separate fetal cells from maternal blood samples.
K. Takahashi et al., J. Nanobiotechnology, 2, 5 (13 Jun. 2004) (6 pp) disclose on-chip cell sorting systems wherein multiple microfluidic inlet passageways lead to a central cell-sorting region fashioned in a PDMS plate (made in a master mold created in photoresist epoxy resin) that is closed by a glass plate. Agar gel electrodes are provided in the PDMS plate which facilitate the separation of undesired cells by the application of electrostatic forces that direct these cells into a parallel, continuous stream of buffer, during their flow through a short, cell-sorting region of confluence. Also disclosed is a post-type filter arrangement for physically trapping large size dust particles.
U.S. Pat. No. 6,454,924 discloses microfluidic devices wherein analyte-containing liquids are caused to flow generally downward past sample surfaces disposed atop upstanding pillars on which capture agents are attached, with the side surfaces of such pillars having been rendered hydrophobic so as to facilitate flow in channels that they define.
Published International Application WO 2004/029221 discloses microfluidic devices that can be used for cell separation, such as separating fetal red blood cells from maternal blood. A sample including the cells is introduced into a microfluidic channel which contains a plurality of obstacles, with the surfaces of the obstacles having binding moieties, e.g., antibodies, suitably coupled thereto, which moieties will bind to cells in the sample. U.S. Pat. No. 6,344,326 discloses microfluidic devices having a plurality of enrichment channels wherein binding elements, such as antibodies, are coupled to crosslinked glass filaments or the like to capture biomolecules of interest. U.S. Pat. No. 5,147,607 teaches the use of devices for carrying out immunoassays, such as sandwich assays, where antibodies are mobilized in microchannels. A recessed area can be provided in the microchannel that contains a group of protrusions which extend upward from the bottom surface of the channel and to which the antibodies are immobilized.
The foregoing, briefly described references provide evidence that there is continuing searching for improved separation methods for isolating cells or other biomaterials from bodily fluids or the like.